JP2022001888A - Flow cytometry system and particle analysis method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a technique capable of efficiently separating particles in a microchip.
SOLUTION: A first detection unit for detecting a first scattered light generated by irradiating a particle flowing through a flow path with a first light beam at a first position of the flow path, and the particle. A second detection unit that detects the second scattered light generated by irradiating the second light beam at a second position different from the first position of the flow path, and the first scattered light and the second scattered light. It has a circuit that reads light waveform information and detection time, and the circuit calculates the difference between the detection time of the first scattered light and the detection time of the second scattered light, whereby the particles are said to have the same. Provided is a flow cytometry system that calculates an arrival time to reach a preparative part communicating with a flow path.
[Selection diagram] Fig. 1

Description

The present technology relates to a particle sorting device and a particle sorting method. More specifically, the present invention relates to a technique for separating and recovering particles based on the results of analysis by an optical method or the like.

Conventionally, an optical measurement method using flow cytometry (flow cytometer) has been used for analysis of biological particles such as cells, microorganisms and liposomes. A flow cytometer is a device that irradiates particles flowing in a flow path formed in a flow cell, a microchip, or the like with light, and detects and analyzes fluorescence or scattered light emitted from each particle.

Some flow cytometers have a function to separate and collect only particles with specific characteristics based on the analysis results, and a device that specifically targets cells is called a "cell sorter". .. As a preparative method for the cell sorter, a droplet charging method for charging and separating droplets containing particles is mainly adopted (see, for example, Patent Document 1). In a droplet charging type device, a fluid discharged from a flow cell or microchip is atomized, a positive (+) or negative (-) charge is applied to the droplet, and the traveling direction is determined by a deflection plate or the like. By changing it, it will be collected in the specified container.

However, the preparative method for forming droplets, such as the droplet charging method, has a problem that it is easily affected by changes in the measurement environment and fluctuations in hydraulic pressure. Therefore, conventionally, a particle sorting device that sorts in a microchip has also been proposed (see Patent Document 2). Since the particle sorting device described in Patent Document 2 sucks and sorts the particles to be collected by the negative pressure suction portion in the microchip, it does not require droplet formation or charging and damages the particles. It is possible to perform sorting at high speed and stably without any problem.

Japanese Unexamined Patent Publication No. 2009-145213 Japanese Unexamined Patent Publication No. 2012-127922

In the conventional particle sorting device, generally, the acquisition operation of the particles to be collected is controlled after a certain time from the detection time by the photodetector. The time from detection to acquisition is preset based on the hydraulic pressure, the distance from the detection position to the sampling position, and the like. However, the control method in which the arrival time is fixed in this way has a problem that the purity and the acquisition rate of the recovered material decrease when the flow rate of the particles fluctuates.

On the other hand, in the apparatus described in Patent Document 1, in order to prevent a decrease in purity due to a fluctuation in the flow rate of the particles, the moving speed is detected for each particle, and the timing of applying a charge to each particle is determined based on the moving speed. I'm in control. However, in the case of the charged droplet method, it is only necessary to determine which droplet each particle belongs to, but in the case of a device that performs sorting within a microchip, in addition to the attributes of each adjacent particle, the fluid mechanical characteristics Need to be considered. Here, the "attribute of the particle" is whether or not the particle is a particle to be sorted, and the "fluid mechanism characteristic" is a backflow generated at the rising edge of the pulse signal of the acquisition operation.

Further, in the charged droplet method, control is performed on droplets, but in a device that performs sorting in a microchip as described in Patent Document 2, it is necessary to control individual particles. .. Furthermore, the path to reach the acquisition position and the factors that affect the arrival of particles differ between the charged droplet method and the method of sorting in a microchip. For the above reasons, the technique described in Patent Document 1 cannot be simply applied to the apparatus described in Patent Document 2 for sorting in a microchip.

Therefore, it is a main object of the present disclosure to provide a particle sorting apparatus and a particle sorting method capable of efficiently sorting particles in a microchip.

The particle sorting device according to the present disclosure irradiates particles flowing through a flow path provided in a microchip with a first light beam at the first position of the flow path, and at the second position of the flow path. A light irradiation unit that irradiates a second light beam, a first light emitted from the particles by irradiating the first light beam, and a second light emitted from the particles by irradiating the second light beam. The light detection unit to be detected, the preparative unit having a pressure change unit that communicates with the flow path and changes the pressure from the actuator drive, and the particles before and after based on the detection time difference between the first light and the second light. It has a time difference for reaching the preparative unit and a preparative control unit that controls the actuator based on the preparative mode.
The second light beam may have the same wavelength as the first light beam.
The pressure changing portion may be provided in the microchip.
The actuator may change the collection destination of the particles to be sorted and the particles not to be sorted based on the determination result of whether or not the particles are to be sorted.
The preparative section may be composed of a suction flow path and a suction section that communicate with the flow path.
The actuator may be able to expand the volume of the suction portion at any timing.
The modification may be based on expanding the volume of the suction section.
A negative pressure may be generated in the suction portion by expanding the volume of the suction portion.
The preparative mode may be selectable by the user.
The preparative mode may be either a purity priority mode in which purity is prioritized or an acquisition rate priority mode in which acquisition rate is prioritized.
The wavelength of the second light beam may be different from that of the first light beam.
It may further have a calculation unit for calculating the time for the particles to reach the preparative unit from the detection time difference between the first light and the second light.
The preparative control unit may control the actuator based on the data detected by the photodetection unit and the time calculated by the calculation unit.
The calculation unit may calculate the arrival time difference at which the particles before and after reach the preparative unit from the detection time difference.
The particles may be bio-related particles.
It may further have a sample liquid introduction flow path into which the sample liquid containing the particles is introduced, and a pair of sheath liquid introduction flow paths into which the sheath liquid is introduced.
The photodetector may be composed of any one or more area image pickup devices selected from the group consisting of a PMT (Photo Multiplier Tube), a CCD, and a CMOS element.
The light detection unit may detect any one or more light selected from the group consisting of forward scattered light, side scattered light, Rayleigh scattered light, and me scattered light.

In the particle sorting system according to the present disclosure, the particles flowing through the flow path provided in the microchip are irradiated with the first light beam at the first position of the flow path, and at the second position of the flow path. A light irradiation unit that irradiates a second light beam, a first light emitted from the particles by irradiating the first light beam, and a second light emitted from the particles by irradiating the second light beam. A particle preparative device having a light detection unit for detection, a preparative unit having a pressure changing unit that communicates with the flow path and changes in pressure from an actuator drive, and the first light and the second light. It has a time difference in which particles before and after the particles reach the preparative unit based on the detection time difference, and a preparative control device for controlling the actuator based on the preparative mode.
Further, in the particle sorting method according to the present disclosure, the particles flowing through the flow path provided in the microchip are irradiated with the first light beam at the first position of the flow path, and the second light beam of the flow path is irradiated. A light irradiation step of irradiating a second light beam at a position, a first light emitted from the particles by irradiating the first light beam, and a second light emitted from the particles by irradiating the second light beam. The light detection step of detecting light, the step based on the preparative section having a pressure changing section that communicates with the flow path and changes the pressure from the actuator drive, and the detection time difference between the first light and the second light. The time difference between the front and rear particles reaching the preparative section and the preparative control step of controlling the actuator based on the preparative mode are performed.

According to the present disclosure, the preparative control unit determines whether or not to collect the particles based on the data of each particle detected by the photodetection unit and the arrival time calculated by the arrival time calculation unit. Therefore, the particle acquisition performance can be improved.

It is a figure which shows typically the structure of the particle sorting apparatus which concerns on 1st Embodiment of this disclosure. It is a figure which shows the detection data by the photodetector 7 shown in FIG. It is a block diagram which shows the circuit structure of the arrival time calculation unit 8 and the preparative control unit 9 of the particle preparative apparatus which concerns on the modification of 1st Embodiment of this disclosure. A and B are diagrams showing processing in the event detection circuit shown in FIG. A and B are diagrams showing the distances in the gating circuit shown in FIG. It is a figure which shows the operation of the acquisition priority mode. A and B are diagrams showing detection data when particles are in close proximity. It is a figure which shows the operation of a purity priority mode.

Hereinafter, embodiments for carrying out the present disclosure will be described in detail with reference to the accompanying drawings. The present disclosure is not limited to each of the following embodiments. The explanations will be given in the following order.

1. 1. First Embodiment (Example of a particle sorting device including a sorting control unit)
2. 2. Modification example of the first embodiment (example of a particle sorting device provided with a mode switching function)

<1. First Embodiment>
First, the particle sorting device according to the first embodiment of the present disclosure will be described. FIG. 1 is a diagram showing a schematic configuration of a particle sorting device according to the first embodiment of the present disclosure. Further, FIG. 2 is a diagram showing detection data by the photodetector 7.

[Overall configuration of device]
As shown in FIG. 1, the particle sorting apparatus of the present embodiment separates and recovers particles 10 based on the results of analysis by an optical method or the like. This particle sorting device includes, for example, a flow path 1, a sorting section 2, an excitation light irradiation section 3, a velocity detection light irradiation section 4, a light detection section 7, an arrival time calculation section 8, a sorting control section 9, and the like. I have.

[About particle 10]
The particles 10 analyzed and fractionated by the particle sorter of the present embodiment widely include biological particles such as cells, microorganisms and ribosomes, or synthetic particles such as latex particles, gel particles and industrial particles. Will be.

Biological particles include chromosomes, ribosomes, mitochondria, organelles (organelles) and the like that make up various cells. In addition, cells include plant cells, animal cells, blood cell lineage cells and the like. Further, the microorganisms include bacteria such as Escherichia coli, viruses such as tobacco mosaic virus, and fungi such as yeast. The bio-related particles may also include bio-related polymers such as nucleic acids, proteins, and complexes thereof.

On the other hand, examples of the industrial particles include those formed of an organic polymer material, an inorganic material, a metal material, or the like. As the organic polymer material, polystyrene, styrene / divinylbenzene, polymethylmethacrylate and the like can be used. Further, as the inorganic material, glass, silica, a magnetic material and the like can be used. As the metal material, for example, gold colloid and aluminum can be used. The shape of these particles is generally spherical, but may be non-spherical, and the size and mass are not particularly limited.

[Flow path 1]
The flow path 1 is formed in the microchip, and a liquid (sample liquid) containing the particles 10 to be sorted is introduced. Here, the microchip provided with the flow path 1 can be formed of glass or various plastics (PP, PC, COP, PDMS, etc.). Further, the material of the microchip has transparency to the light emitted from the excitation light irradiation unit 3 and the speed detection light irradiation unit 4, has less autofluorescence, and has less optical error because the wavelength dispersion is small. It is desirable to use the material.

On the other hand, the flow path 1 can be formed by wet etching or dry etching of a glass substrate, or by nanoimprinting, injection molding, or machining of a plastic substrate. Then, the microchip can be formed by sealing, for example, a substrate on which a flow path 1 or the like is formed with a substrate of the same material or a different material.

Note that FIG. 1 shows only the portion of the flow path 1 that is irradiated with the excitation light or the velocity detection light, but the sample liquid introduction flow in which the sample liquid containing the particles 10 is introduced upstream of this. A path and a pair of sheath liquid introduction channels into which the sheath liquid is introduced may be provided. In this case, the sheath liquid introduction flow path merges with the sample liquid introduction flow path from both sides, and the flow path 1 is provided on the downstream side of the confluence point. Then, in the flow path 1, the sample flow is surrounded by a sheath flow, and the liquid flows in a state where a laminar flow is formed, and the particles 10 in the sample liquid are arranged in substantially one row with respect to the flow direction. Flow side by side.

[Preparation section 2]
The sorting unit 2 sorts the particles 10 to be collected and is formed in the microchip. The preparative section 2 communicates with the downstream end portion of the flow path 1 and is composed of a suction flow path 21, a negative pressure suction section 22, and the like. The structure of the negative pressure suction unit 22 is not particularly limited as long as it can suck the fine particles to be collected at a predetermined timing, but the negative pressure suction unit 22 is, for example, by an actuator (not shown) or the like. The volume of 22 can be expanded at any time.

[Excitation light irradiation unit 3]
The excitation light irradiation unit 3 is provided with a light source 31 that generates excitation light such as laser light, an optical system 32 that forms a spot shape, a mirror 33, and the like. Then, for example, the particles 10 passing through the flow path 1 formed in the microchip are irradiated with the excitation light. Although FIG. 1 shows an example of the case where one light source 31 is used, the present disclosure is not limited to this, and two or more light sources 31 may be provided. In that case, each light source 31 may be provided. Light of different wavelengths may be emitted from the light source 31.

[Light irradiation unit for speed detection 4]
The speed detection light irradiation unit 4 is provided with a light source 41 that generates speed detection light, an optical system 42 that forms a spot shape, a mirror 43, and the like. Then, for example, the particles 10 flowing through the flow path 1 formed in the microchip are irradiated with the velocity detection light at a position different from the excitation light described above. The speed detection light may be light having the same wavelength as the excitation light, but from the viewpoint of simplifying the device configuration, it is preferable to use light having a wavelength different from that of the excitation light.

[Light detection unit 7]
The light detection unit 7 detects light (scattered light, fluorescence, etc.) generated from the particles 10 passing through the flow path 1, and is a 0th-order light removing member 71, mirrors 72a to 72d, and photodetectors 73a to. It is composed of 73d and the like. For the photodetectors 73a to 73d, for example, a PMT (Photo Multiplier Tube) or an area image pickup element such as a CCD or CMOS element can be used.

In the photodetector 7, for example, the photodetector 73a emits the forward scattered light derived from the excitation light, the photodetector 73b emits the scattered light derived from the velocity detection light, and the photodetectors 73c and 73d emit fluorescence, respectively. To detect. The light to be detected by the photodetector 7 is not limited to these, and may detect lateral scattered light, Rayleigh scattering, Mie scattering, and the like. Then, the light detected by the photodetector 7 is converted into an electric signal.

[Arrival time calculation unit 8]
From the detection time difference between the light derived from the excitation light and the light derived from the velocity detection light, the time for each particle 10 to reach the preparative unit 2 communicating with the flow path is individually calculated. The method for calculating the arrival time is not particularly limited, but for example, as shown in FIG. 2, the forward scattered light (Ch1 data) derived from the excitation light detected by the light detection unit 7 and the speed detection. The arrival time of each particle 10 is calculated from the detection time difference of the forward scattered light (Ch2 data) derived from the light.

Here, the arrival time to the preparative unit 2 can be calculated by, for example, a simple linear approximation formula shown in the following formula 1. In the following formula 1, L1 is the distance between the excitation light irradiation position and the speed detection light irradiation position, and L2 is the distance from the speed detection light irradiation position to the suction flow path 21 of the preparative unit 2 (see FIG. 1). ). Further, T1 in the following mathematical formula 1 is the detection time of the light derived from the excitation light, T2 is the detection time of the light derived from the velocity detection light, and (T1-T2) is the difference between these detection times (FIG. 2).

The method of calculating the arrival time to the preparative unit 2 is not limited to the linear calculation method shown in the above equation 1, and other calculation methods such as polynomial approximation and look-up table may be used.

[Preparation control unit 9]
The preparative control unit 9 controls the preparative of the particles 10, and the particles are based on the data of each particle 10 detected by the light detection unit 7 and the arrival time calculated by the arrival time calculation unit 8. Determine whether or not to collect 10. The preparative control unit 9 calculates, for example, the arrival time difference between the particles 10 before and after, and determines that the particles whose calculated arrival time difference is equal to or less than a preset threshold value are “non-collected”. As a result, when the particles 10 are flowing in close proximity to each other, it is possible to prevent the particles before and after the particles to be collected from being caught and acquired.

Further, the preparative control unit 9 controls the timing of collecting the particles 10 by the preparative unit 2 by, for example, controlling the operation of the negative pressure suction unit 22 based on the above-mentioned determination result. This makes it possible to improve the acquisition accuracy of the target particles and perform fractionation with high purity and acquisition rate.

[motion]
Next, the operation of the particle sorting device of this embodiment will be described. When the particles are sorted by the particle sorting device of the present embodiment, the sample liquid containing the particles to be sorted is introduced into the sample inlet provided in the microchip, and the sheath liquid is introduced into the sheath inlet. .. Then, the particles 10 passing through the flow path 1 are irradiated with the excitation light, and the particles 10 are irradiated with the velocity detection light at a position different from the excitation light. At this time, as shown in FIG. 1, the excitation light and the speed detection light may be focused by one condenser lens 5 and irradiated to the particles 10, but they are condensed by different condenser lenses. May be good.

Next, the detection unit 7 detects the light emitted from each particle 10, and the arrival time calculation unit 8 collects the light from the detection time difference between the light derived from the excitation light and the light derived from the velocity detection light. The time for each particle 10 to reach 2 is calculated individually. At this time, as shown in FIG. 1, the light derived from the excitation light and the light derived from the speed detection light are collected by one condenser lens 6 and collected by the 0th-order light removing member 71 of the detection unit 7. It may be illuminated, but it may be condensed by different condenser lenses.

After that, in the preparative control unit 9, whether to collect the particles 10 from the optical characteristic data of each particle 10 detected by the detection unit 7 and the arrival time to the preparative unit 2 calculated by the arrival time calculation unit 8. Judge whether or not. Then, based on the determination result, the preparative control unit 9 controls the timing of collecting the particles 10 in the preparative unit 2. For example, when the preparative unit 2 has a negative pressure suction unit 22 communicating with the flow path 1, the preparative control unit 9 controls the operation of an actuator or the like provided in the negative pressure suction unit 22.

As described in detail above, in the particle sorting apparatus of the present embodiment, the arrival time to the sorting section is calculated for each particle, and not only the optical characteristic data of each particle but also the arrival time to the sorting section is calculated. Also, it is decided whether or not to collect the particles. This makes it possible to separate the particles with high purity or high acquisition rate regardless of the flow position and flow state of the particles. As a result, the acquisition performance can be improved as compared with the conventional particle sorting device.

Further, since the particle sorting device of the present embodiment calculates the arrival time for each particle, it is not easily affected by the change in the flow rate due to the change in the environmental temperature or the remaining amount of the supply tank. This eliminates the need for highly accurate flow control, so low-cost pressure control devices can be adopted, streamline component management and assembly accuracy management can be simplified, and manufacturing costs can be reduced. be able to.

<2. Modification example of the first embodiment>
Next, the particle sorting device according to the modified example of the first embodiment of the present disclosure will be described. In the particle sorting device of this modification, the user can select whether to prioritize "purity" or "acquisition rate" when deciding whether or not to collect the particles.

FIG. 3 is a block diagram showing a circuit configuration of an arrival time calculation unit 8 and a preparative control unit 9 of the particle preparative device of this modification. Further, FIGS. 4A and 4B are diagrams showing processing in the event detection circuit, and FIGS. 5A and 5B are diagrams showing distances in the gating circuit. Sorting in the "purity priority mode" or the "acquisition rate priority mode" can be realized by, for example, the circuit having the configuration shown in FIG.

[Event detection circuit]
The event detection circuit triggers with the detection signals of Ch1 and Ch2 to read the waveform of each Ch, and calculates the width, height, and area shown in FIG. 4A. Then, for the Ch1 detection data regarding the forward scattered light derived from the excitation light and the Ch2 detection data regarding the forward scattered light derived from the velocity detection light, the time at the center of the waveform is calculated and used as the detection time.

Then, as shown in FIG. 4B, in the event detection circuit, for each particle 10, Ch1 (forward scattered light derived from excitation light) and Ch2 (forward scattered light derived from velocity detection light) acquired in time series. The detection signal of each particle is associated and the detection data (event) of each particle is packetized. The packet contains an item that is updated as the subsequent processing progresses, and the Flag is basically 1/0, which corresponds to acquisition / non-acquisition determined by each logic. As the detection time, the trigger time of Ch1 and Ch2 can also be used.

[Arrival time calculation circuit]
The arrival time calculation circuit uses the detection times (T1 and T2) of Ch1 and Ch2 to calculate the arrival time from the above equation 1 and the like, and sets it as the "Sorting Time" of the event packet.

[Gating circuit]
The gating circuit determines "acquisition / non-acquisition" of the particle 10 based on a preset threshold value, and sets the "Gate Flag" of the event packet. For example, before the start of the gating acquisition operation, a histogram chart shown in FIG. 5A, a 2D chart shown in FIG. 5B, and the like are plotted on a GUI on a control computer, and a group of particles to be acquired (having the desired characteristics). (Particle group) is specified by enclosing it in a geometric shape, for example.

The parameter (threshold value) for determining "acquisition / non-acquisition" may be any of the width, height, and area of the detection data acquired in each Ch, and these may be combined.

[Output queue circuit]
The output queue circuit sorts the detection data (events) of each particle 10 in the order of arrival at the preparative unit based on the arrival time at the preparative unit (“Sorting Time”). After that, "acquisition / non-acquisition" is determined according to the preparative mode selected by the user such as "purity priority" or "acquisition rate priority". Then, based on the result, "Sort Flag" is set.

When the particles 10 are flowing in close proximity to each other, the front and rear particles 10 may be involved in one acquisition operation, and the plurality of particles 10 may be collected in the preparative unit 2. The "purity priority mode" and the "acquisition rate priority mode" differ in the method of determining "acquisition / non-acquisition" when the particles 10 are in close proximity to each other. FIG. 6 is a diagram showing the operation of the acquisition priority mode. Further, FIGS. 7A and 7B are diagrams showing detection data when particles are in close proximity to each other. Further, FIG. 8 is a diagram showing the operation of the purity priority mode.

The "acquisition rate priority mode" is a mode in which the number of acquired particles is increased even if the purity of the captured particles decreases, and as shown in FIG. 6, even when the particles 10 are flowing in close proximity to each other, the particles are to be sorted. Collect the particles. On the other hand, the "purity priority mode" is a mode for increasing the purity of the captured particles, and when the acquired particles and the non-acquired particles come close to each other, the acquired particles are intentionally captured in order to prevent them from being captured together. Is judged as "non-acquisition".

In particular, in the "purity priority mode", as shown in FIG. 8, when the detection data (event) of the particle 10 detected later is close to the previous particle 10, the "acquisition / non-acquisition" of the previous event is performed. "Acquisition" also needs to be judged again. Here, ΔT1 shown in FIG. 7A is a set value and is the time for entraining the next particle (T1 = Tn + ΔT1). Further, ΔT2 is also a set value, which is the time for entraining the previous particle (T2 = Tn + ΔT2).

[Output timing generation circuit]
The output timing generation circuit reads the time (Sorting time) of the event to be acquired first in the output queue, compares it with the Clock Counter value, and generates an output timing signal at that time.

[Output signal generation circuit]
The output signal generation circuit detects the output timing signal and outputs a waveform signal that controls the actuation device of the preparative unit 2.

The purpose of the particle sorting device of this modification is that the user can select whether to prioritize "purity" or "acquisition rate" when deciding whether to collect the particles. It is possible to sort according to the amount. The configurations and effects other than the above in this modification are the same as those in the first embodiment described above.

In addition, the present disclosure may have the following structure.
(1)
An excitation light irradiation unit that irradiates particles flowing through the flow path with excitation light,
A velocity detection light irradiation unit that irradiates the particles with velocity detection light at a position different from the excitation light.
A photodetector that detects the light emitted from the particles,
An arrival time calculation unit that individually calculates the time for each particle to reach the preparative unit communicating with the flow path from the detection time difference between the light derived from the excitation light and the light derived from the velocity detection light.
It has a preparative control unit that controls the preparative of the particles.
The flow path and the preparative portion are provided in the microchip, and the flow path and the preparative portion are provided in the microchip.
The preparative control unit determines whether or not to collect the particles based on the data of each particle detected by the light detection unit and the arrival time calculated by the arrival time calculation unit. Device.
(2)
The particle sorting device according to (2), wherein the preparative control unit calculates the arrival time difference between the front and rear particles, and determines that the particles whose arrival time difference is equal to or less than the threshold value are not collected.
(3)
The particle sorting device according to (1) or (2), wherein the velocity detection light has a wavelength different from that of the excitation light.
(4)
The particle sorting device according to (3), wherein the arrival time calculation unit calculates the arrival time of each particle from the detection time difference between the scattered light derived from the excitation light and the scattered light derived from the velocity detection light.
(5)
The particle sorting device according to any one of (1) to (4), wherein the excitation light irradiation unit includes two or more light sources that emit light having different wavelengths.
(6)
The particle sorting device according to any one of (1) to (5), wherein the sorting unit has a negative pressure suction unit communicating with the flow path.
(7)
The preparative control unit controls the operation of the negative pressure suction unit based on the data of each particle detected by the light detection unit and the arrival time calculated by the arrival time calculation unit (6). The described particle sorter.
(8)
The preparative control unit controls the timing of collecting the particles in the preparative unit based on the data of each particle detected by the light detection unit and the arrival time calculated by the arrival time calculation unit. The particle sorting device according to any one of (1) to (7).
(9)
An excitation light irradiation step of irradiating particles flowing through a flow path provided in a microchip with excitation light,
A velocity detection light irradiation step of irradiating the particles with velocity detection light at a position different from the excitation light,
A photodetection step that detects the light emitted from the particles, and
From the detection time difference between the light derived from the excitation light and the light derived from the velocity detection light, the time for each particle to reach the preparative portion provided in the microchip and communicating with the flow path is individually determined. The arrival time calculation process to be calculated and
A preparative control step for determining whether or not to collect the particles based on the data of each particle detected in the light detection step and the arrival time calculated in the arrival time calculation step.
Particle sorting method.
(10)
The particle sorting method according to (9), wherein the preparative control step calculates the arrival time difference between the particles before and after, and determines that the particles whose arrival time difference is equal to or less than the threshold value are not collected.
(11)
The particle sorting method according to (9) or (10), wherein light having a wavelength different from that of the excitation light is used as the velocity detection light.
(12)
The particle fractionation method according to (11), wherein the arrival time calculation step calculates the arrival time of each particle from the detection time difference between the scattered light derived from the excitation light and the scattered light derived from the velocity detection light.
(13)
The particle fractionation method according to any one of (9) to (12), wherein the excitation light irradiation step emits light having a different wavelength from two or more light sources.
(14)
The preparative section has a negative pressure suction section that communicates with the flow path.
The preparative control step controls the operation of the negative pressure suction unit based on the data of each particle detected in the light detection step and the arrival time calculated in the arrival time calculation step (9) to (13). ). The particle sorting method according to any one of.
(15)
The preparative control step controls the timing of collecting the particles in the preparative unit based on the data of each particle detected in the light detection step and the arrival time calculated in the arrival time calculation step (9). )-(14). The particle sorting method according to any one of (14).

1 Channel 2 Preparative unit 3 Excitation light irradiation unit 4 Speed detection light irradiation unit 5, 6 Objective lens 7 Photodetector 8 Arrival time calculation unit 9 Preparative control unit 10 Particles 21 Suction flow path 22 Negative pressure suction unit 31 , 41 Light source 32, 42 Optical system 33, 43, 72a to 72d Mirror 71 10th order light removal member 73a to 73d Photodetector

Claims (16)

A first detection unit that detects the first scattered light generated by irradiating the particles flowing through the flow path with the first light beam at the first position of the flow path.
A second detection unit that detects the second scattered light generated by irradiating the particles with a second light beam at a second position different from the first position of the flow path.
A circuit that reads the waveform information and detection time of the first scattered light and the second scattered light,
Have,
The circuit calculates the arrival time at which the particles reach the preparative portion communicating with the flow path by calculating the difference between the detection time of the first scattered light and the detection time of the second scattered light. , Flow cytometry system. The flow cytometry system according to claim 1, wherein the velocity of the particles is determined based on the difference in arrival times. The flow cytometry system according to claim 1 or 2, wherein the circuit controls the sorting timing by the sorting unit based on the difference in arrival time. The circuit packets the data about the particles and
The flow cytometry system according to any one of claims 1 to 3, wherein the data relating to the particles include waveform information of the first scattered light, waveform information of the second scattered light, and the arrival time. .. The data relating to the particles include any one or more selected from the group consisting of the detection time of the first scattered light, the detection time of the second scattered light, and the sorting timing by the sorting unit. Item 4. The flow cytometry system according to Item 4. The flow cytometry system according to claim 5, wherein the data relating to the particles include any one or more selected from the group consisting of event numbers, trigger channels, trigger times, gate flags, and sort flags. The flow cytometry system according to any one of claims 4 to 6, wherein the circuit outputs data regarding the packetized particles. Any one of claims 1 to 7, wherein the first scattered light and the second scattered light are one or more of the group consisting of forward scattered light, side scattered light, Rayleigh scattering, and me scattering. Flow cytometry system as described in section. The flow cytometry system according to any one of claims 1 to 8, wherein the circuit associates each channel information with the information of each scattered light. The flow cytometry system according to claim 9, wherein the circuit associates each channel information with the detection time of each scattered light. The first irradiation unit that irradiates the first light beam and
The second irradiation unit that irradiates the second light beam and
The flow cytometry system according to any one of claims 1 to 10, further comprising. The flow cytometry system according to any one of claims 1 to 11, wherein the wavelength of the first light beam is different from the wavelength of the second light beam. The flow cytometry system according to any one of claims 1 to 12, wherein the inside of the flow path is surrounded by a sheath flow and the liquid flows in a state where a laminar flow is formed. The flow cytometry system according to any one of claims 1 to 13, wherein the waveform information is one or more of the group consisting of width, height, and area. The flow cytometry system according to any one of claims 1 to 14, wherein the detection time is the time at the center of the waveform of each scattered light. The first detection step of detecting the first scattered light generated by irradiating the particles flowing through the flow path with the first light beam at the first position of the flow path, and
A second detection step of detecting the second scattered light generated by irradiating the particles with a second light beam at a second position different from the first position of the flow path.
The step of reading the waveform information and the detection time of the first scattered light and the second scattered light,
A particle analysis method.

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